Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

Digital Methods for Performing Daily Tasks Among Older Adults: An Initial Report of Frequency of Use and Perceived Utility.

OBJECTIVE: Digital technologies permit new ways of performing instrumental activities of daily living (iADLs) for older adults, but these approaches are not usually considered in existing iADL measures. The current study investigated how a sample of older adults report using digital versus analog approaches for iADLs. METHOD: 248 older adults completed the Digital and Analog Daily Activities Survey, a newly developed measure of how an individual performs financial, navigation, medication, and other iADLs. RESULTS: The majority of participants reported regularly using digital methods for some iADLs, such as paying bills (67.7%) and using GPS (67.7%). Low digital adopters were older than high adopters (F(2, 245) = 12.24, p < .001), but otherwise the groups did not differ in terms of gender, years of education, or history of neurological disorders. Participants who used digital methods relatively more than analog methods reported greater levels of satisfaction with their approach and fewer daily errors. CONCLUSIONS: Many older adults have adopted digital technologies for supporting daily tasks, which suggests limitations to the validity of current iADL assessments. By capitalizing on existing habits and enriching environments with new technologies, there are opportunities to promote technological reserve in older adults in a manner that sustains daily functioning.

A community genomics approach to natural hybridization.

Hybridization is a complicated, oft-misunderstood process. Once deemed unnatural and uncommon, hybridization is now recognized as ubiquitous among species. But hybridization rates within and among communities are poorly understood despite the relevance to ecology, evolution and conservation. To clarify, we examined hybridization across 75 freshwater fish communities within the Ozarks of the North American Interior Highlands (USA) by single nucleotide polymorphism (SNP) genotyping 33 species (N = 2865 individuals; double-digest restriction site-associated DNA sequencing (ddRAD)). We found evidence of hybridization (70 putative hybrids; 2.4% of individuals) among 18 species-pairs involving 73% (24/33) of study species, with the majority being concentrated within one family (Leuciscidae/minnows; 15 species; 66 hybrids). Interspecific genetic exchange-or introgression-was evident from 24 backcrossed individuals (10/18 species-pairs). Hybrids occurred within 42 of 75 communities (56%). Four selected environmental variables (species richness, protected area extent, precipitation (May and annually)) exhibited 73-78% accuracy in predicting hybrid occurrence via random forest classification. Our community-level assessment identified hybridization as spatially widespread and environmentally dependent (albeit predominantly within one diverse, omnipresent family). Our approach provides a more holistic survey of natural hybridization by testing a wide range of species-pairs, thus contrasting with more conventional evaluations.

Riverscape community genomics: A comparative analytical approach to identify common drivers of spatial structure.

Genetic differentiation among local groups of individuals, that is, genetic ß-diversity, is a key component of population persistence related to connectivity and isolation. However, most genetic investigations of natural populations focus on a single species, overlooking opportunities for multispecies conservation plans to benefit entire communities in an ecosystem. We present an approach to evaluate genetic ß-diversity within and among many species and demonstrate how this riverscape community genomics approach can be applied to identify common drivers of genetic structure. Our study evaluated genetic ß-diversity in 31 co-distributed native stream fishes sampled from 75 sites across the White River Basin (Ozarks, USA) using SNP genotyping (ddRAD). Despite variance among species in the degree of genetic divergence, general spatial patterns were identified corresponding to river network architecture. Most species (N = 24) were partitioned into discrete subpopulations (K = 2-7). We used partial redundancy analysis to compare species-specific genetic ß-diversity across four models of genetic structure: Isolation by distance (IBD), isolation by barrier (IBB), isolation by stream hierarchy (IBH), and isolation by environment (IBE). A significant proportion of intraspecific genetic variation was explained by IBH (x̄ = 62%), with the remaining models generally redundant. We found evidence for consistent spatial modularity in that gene flow is higher within rather than between hierarchical units (i.e., catchments, watersheds, basins), supporting the generalization of the stream hierarchy model. We discuss our conclusions regarding conservation and management and identify the 8-digit hydrologic unit (HUC) as the most relevant spatial scale for managing genetic diversity across riverine networks.

Private Payer and Medicare Coverage Policies for Use of Circulating Tumor DNA Tests in Cancer Diagnostics and Treatment.

BACKGROUND: Circulating tumor DNA (ctDNA) is used to select initial targeted therapy, identify mechanisms of therapeutic resistance, and measure minimal residual disease (MRD) after treatment. Our objective was to review private and Medicare coverage policies for ctDNA testing. METHODS: Policy Reporter was used to identify coverage policies (as of February 2022) from private payers and Medicare Local Coverage Determinations (LCDs) for ctDNA tests. We abstracted data regarding policy existence, ctDNA test coverage, cancer types covered, and clinical indications. Descriptive analyses were performed by payer, clinical indication, and cancer type. RESULTS: A total of 71 of 1,066 total policies met study inclusion criteria, of which 57 were private policies and 14 were Medicare LCDs; 70% of private policies and 100% of Medicare LCDs covered at least one indication. Among 57 private policies, 89% specified a policy for at least 1 clinical indication, with coverage for ctDNA for initial treatment selection most common (69%). Of 40 policies addressing progression, coverage was provided 28% of the time, and of 20 policies addressing MRD, coverage was provided 65% of the time. Non-small cell lung cancer (NSCLC) was the cancer type most frequently covered for initial treatment (47%) and progression (60%). Among policies with ctDNA coverage, coverage was restricted to patients without available tissue or in whom biopsy was contraindicated in 91% of policies. MRD was commonly covered for hematologic malignancies (30%) and NSCLC (25%). Of the 14 Medicare LCD policies, 64% provided coverage for initial treatment selection and progression, and 36% for MRD. CONCLUSIONS: Some private payers and Medicare LCDs provide coverage for ctDNA testing. Private payers frequently cover testing for initial treatment, especially for NSCLC, when tissue is insufficient or biopsy is contraindicated. Coverage remains variable across payers, clinical indications, and cancer types despite inclusion in clinical guidelines, which could impact delivery of effective cancer care.

The Health Inequality Impact of Liquid Biopsy to Inform First-Line Treatment of Advanced Non-Small Cell Lung Cancer: A Distributional Cost-Effectiveness Analysis.

OBJECTIVES: To perform a distributional cost-effectiveness analysis of liquid biopsy (LB) followed by, if needed, tissue biopsy (TB) (LB-first strategy) relative to a TB-only strategy to inform first-line treatment of advanced non-small cell lung cancer (aNSCLC) from a US payer perspective by which we quantify the impact of LB-first on population health inequality according to race and ethnicity. METHODS: With a health economic model, quality-adjusted life-years (QALYs) and costs per patient were estimated for each subgroup. Given the lifetime risk of aNSCLC, and assuming equally distributed opportunity costs, the incremental net health benefits of LB-first were calculated, which were used to estimate general population quality-adjusted life expectancy at birth (QALE) by race and ethnicity with and without LB-first. The degree of QALYs and QALE differences with the strategies was expressed with inequality indices. Their differences were defined as the inequality impact of LB-first. RESULTS: LB-first resulted in an additional 0.21 (95% uncertainty interval: 0.07-0.39) QALYs among treated patients, with the greatest gain observed among Asian patients (0.31 QALYs [0.09-0.61]). LB-first resulted in an increase in relative inequality in QALYs among patients, but a minor decrease in relative inequality in QALE. CONCLUSIONS: LB-first to inform first-line aNSCLC therapy can improve health outcomes. With current diagnostic performance, the benefit is the greatest among Asian patients, thereby potentially widening racial and ethnic differences in survival among patients with aNSCLC. Assuming equally distributed opportunity costs and access, LB-first does not worsen and, in fact, may reduce inequality in general population health according to race and ethnicity.

Piecemeal stewardship activities miss numerous social and environmental benefits associated with culturally appropriate ways of caring for country.

Prior research has identified both the contribution that people make to nature and the contribution that nature makes to people (by enhancing wellbeing) - with clear conceptual models to describe the interactions. Prior research has also made a clear case for incorporating insights from multiple perspectives and knowledge systems when seeking to better understand this interactive system. What is lacking, is guidance on how to operationalise some of these ideas to provide bespoke advice to environmental managers. Arguably, we have an adequate, albeit imperfect, understanding of how to operationalise (measure, value and/or otherwise account for) some parts of the conceptual model. There is, for example, abundant literature that describes different ways of valuing Ecosystem services, and a growing body of literature that describes and quantifies the ecological benefits of various stewardship activities, which will subsequently also generate an indirect benefit to people (since improved ecological conditions will improve Ecosystem services). In comparison, we know relatively little about the way in which stewardship activities directly benefit people - and it is on this gap that our paper focuses. We partially fill that knowledge gap by first reaching out to and learning from some of Australia's First Nations People. Key learnings underscore the inter-connectedness of the system, and the need for resource managers to not only monitor the extent and condition of natural system but also the extent and condition of an inextricably connected human system, in addition to the human-nature interactions. We clearly identify ways in which those insights can be used to improve and extend accounting frameworks, such as SEEA Ecosystem Accounts developed by the United Nations that are often used by natural resource managers. In so doing, we generate new insights about Indigenous stewardship (Caring for Country) and methods of accounting for and monitoring stewardship activities. As such, our work provides a practical illustration of one way to populate conceptual models with 'real world' data that also incorporates different world views, to support decision makers for improved social and environmental outcomes.

US private payers' perspectives on insurance coverage for genome sequencing versus exome sequencing: A study by the Clinical Sequencing Evidence-Generating Research Consortium (CSER).

PURPOSE: There is limited payer coverage for genome sequencing (GS) relative to exome sequencing (ES) in the U.S. Our objective was to assess payers' considerations for coverage of GS versus coverage of ES and requirements payers have for coverage of GS. The study was conducted by the NIH-funded Clinical Sequencing Evidence-Generating Research Consortium (CSER). METHODS: We conducted semi-structured interviews with representatives of private payer organizations (payers, N = 12) on considerations and evidentiary and other needs for coverage of GS and ES. Data were analyzed using thematic analysis. RESULTS: We described four categories of findings and solutions: demonstrated merits of GS versus ES, enhanced methods for evidence generation, consistent laboratory processes/sequencing methods, and enhanced implementation/care delivery. Payers see advantages to GS vs. ES and are open to broader GS coverage but need more proof of these advantages to consider them in coverage decision-making. Next steps include establishing evidence of benefits in specific clinical scenarios, developing quality standards, ensuring transparency of laboratory methods, developing clinical centers of excellence, and incorporating the role of genetic professionals. CONCLUSION: By comparing coverage considerations for GS and ES, we identified a path forward for coverage of GS. Future research should explicitly address payers' conditions for coverage.

Environmental drivers of recruitment in a tropical fishery: Monsoonal effects and vulnerability to water abstraction.

Fisheries and natural water resources across the world are under increasing pressure from human activity, including fishing and irrigated agriculture. There is an urgent need for information on the climatic/hydrologic drivers of fishery productivity that can be readily applied to management. We use a generalized linear mixed model framework of catch curve regression to resolve the key climatic/hydrological drivers of recruitment in Barramundi Lates calcarifer using biochronological (otolith aging) data collected from four river-estuary systems in the Northern Territory, Australia. These models were then used to generate estimates of the year class strength (YCS) outcomes of different water abstraction scenarios (ranging from 10% to 40% abstraction per season/annum) for two of the rivers in low, moderate, and high discharge years. Barramundi YCS displayed strong interannual variation and was positively correlated with regional monsoon activity in all four rivers. River-specific analyses identified strong relationships between YCS and several river-specific hydrology variables, including wet and dry season discharge and flow duration. Water abstraction scenario models based on YCS-hydrology relationships predicted reductions of >30% in YCS in several cases, suggesting that increased water resource development in the future may pose risks for Barramundi recruitment and fishery productivity. Our study demonstrates the importance of the tropical monsoon as a driver of Barramundi recruitment and the potential for detrimental impacts of increased water abstraction on fishery productivity. The biochronological and statistical approaches we used have the potential to be broadly applied to inform policy and management of water resource and fisheries.

Influence of payer coverage and out-of-pocket costs on ordering of NGS panel tests for hereditary cancer in diverse settings.

The landscape of payment for genetic testing has been changing, with an increase in the number of laboratories offering testing, larger panel offerings, and lower prices. To determine the influence of payer coverage and out-of-pocket costs on the ordering of NGS panel tests for hereditary cancer in diverse settings, we conducted semi-structured interviews with providers who conduct genetic counseling and order next-generation sequencing (NGS) panels purposefully recruited from 11 safety-net clinics and academic medical centers (AMCs) in California and North Carolina, states with diverse populations and divergent Medicaid expansion policies. Thematic analysis was done to identify themes related to the impact of reimbursement and out-of-pocket expenses on test ordering. Specific focus was put on differences between settings. Respondents from both safety-net clinics and AMCs reported that they are increasingly ordering panels instead of single-gene tests, and tests were ordered primarily from a few commercial laboratories. Surprisingly, safety-net clinics reported few barriers to testing related to cost, largely due to laboratory assistance with prior authorization requests and patient payment assistance programs that result in little to no patient out-of-pocket expenses. AMCs reported greater challenges navigating insurance issues, particularly prior authorization. Both groups cited non-coverage of genetic counseling as a major barrier to testing. Difficulty of access to cascade testing, particularly for family members that do not live in the United States, was also of concern. Long-term sustainability of laboratory payment assistance programs was a major concern; safety-net clinics were particularly concerned about access to testing without such programs. There were few differences between states. In conclusion, the use of laboratories with payment assistance programs reduces barriers to NGS panel testing among diverse populations. Such programs represent a major change to the financing and affordability of genetic testing. However, access to genetic counseling is a barrier and must be addressed to ensure equity in testing.

Genetic counselors' experience with reimbursement and patient out-of-pocket cost for multi-cancer gene panel testing for hereditary cancer syndromes.

Multi-cancer gene panels for hereditary cancer syndromes (hereditary cancer panels, HCPs) are widely available, and some laboratories have programs that limit patients' out-of-pocket (OOP) cost share. However, little is known about practices by cancer genetic counselors for discussing and ordering an HCP and how insurance reimbursement and patient out-of-pocket share impact these practices. We conducted a survey of cancer genetic counselors based in the United States through the National Society of Genetic Counselors to assess the impact of reimbursement and patient OOP share on ordering of an HCP and hereditary cancer genetic counseling. Data analyses were conducted using chi-square and t tests. We received 135 responses (16% response rate). We found that the vast majority of respondents (94%, 127/135) ordered an HCP for patients rather than single-gene tests to assess hereditary cancer predisposition. Two-thirds of respondents reported that their institution had no protocol related to discussing HCPs with patients. Most respondents (84%, 114/135) indicated clinical indications and patients' requests as important in selecting and ordering HCPs, while 42%, 57/135, considered reimbursement and patient OOP share factors important. We found statistically significant differences in reporting of insurance as a frequently used payment method for HCPs and in-person genetic counseling (84% versus 59%, respectively, p < 0.0001). Perceived patient willingness to pay more than $100 was significantly higher for HCPs than for genetic counseling(41% versus 22%, respectively, p < 0.01). In sum, genetic counselors' widespread selection and ordering of HCPs is driven more by clinical indications and patient preferences than payment considerations. Respondents perceived that testing is more often reimbursed by insurance than genetic counseling, and patients are more willing to pay for an HCP than for genetic counseling. Policy efforts should address this incongruence in reimbursement and patient OOP share. Patient-centered communication should educate patients on the benefit of genetic counseling.

ClineHelpR: an R package for genomic cline outlier detection and visualization.

BACKGROUND: Patterns of multi-locus differentiation (i.e., genomic clines) often extend broadly across hybrid zones and their quantification can help diagnose how species boundaries are shaped by adaptive processes, both intrinsic and extrinsic. In this sense, the transitioning of loci across admixed individuals can be contrasted as a function of the genome-wide trend, in turn allowing an expansion of clinal theory across a much wider array of biodiversity. However, computational tools that serve to interpret and consequently visualize 'genomic clines' are limited, and users must often write custom, relatively complex code to do so. RESULTS: Here, we introduce the ClineHelpR R-package for visualizing genomic clines and detecting outlier loci using output generated by two popular software packages, bgc and Introgress. ClineHelpR bundles both input generation (i.e., filtering datasets and creating specialized file formats) and output processing (e.g., MCMC thinning and burn-in) with functions that directly facilitate interpretation and hypothesis testing. Tools are also provided for post-hoc analyses that interface with external packages such as ENMeval and RIdeogram. CONCLUSIONS: Our package increases the reproducibility and accessibility of genomic cline methods, thus allowing an expanded user base and promoting these methods as mechanisms to address diverse evolutionary questions in both model and non-model organisms. Furthermore, the ClineHelpR extended functionality can evaluate genomic clines in the context of spatial and environmental features, allowing users to explore underlying processes potentially contributing to the observed patterns and helping facilitate effective conservation management strategies.

Private payer coverage policies for ApoE-e4 genetic testing.

PURPOSE: ApoE-e4 has a well-established connection to late-onset Alzheimer disease (AD) and is available clinically. Yet, there have been no analyses of payer coverage policies for ApoE. Our objective was to analyze private payer coverage policies for ApoE genetic testing, examine the rationales, and describe supporting evidence referenced by policies. METHODS: We searched for policies from the eight largest private payers (by member numbers) covering ApoE testing for late-onset AD. We implemented content analysis methods to evaluate policies for coverage decisions and rationales. RESULTS: Seven payers had policies with positions on ApoE testing. Five explicitly state they do not cover ApoE and two apply generic preauthorization criteria. Rationales supporting coverage decisions include: reference to guidelines or national standards, inadequate data supporting testing, characterizing testing as investigational, or that testing would not alter patients' clinical management. CONCLUSION: Seven of the eight largest private payers' coverage policies reflect standards that discourage ApoE testing due to a lack of clinical utility. As the field advances, ApoE testing may have an important clinical role, particularly considering that disease-modifying therapies are under evaluation by the US Food and Drug Administration. These types of field advancements may not be consistent with private payers' policies and may cause payers to reevaluate existing coverage policies.

Laboratory business models and practices: implications for availability and access to germline genetic testing.

PURPOSE: Germline testing laboratories have evolved over several decades. We describe laboratory business models and practices and explore their implications on germline testing availability and access. METHODS: We conducted semistructured interviews with key informants using purposive sampling. We interviewed 13 key informants representing 14 laboratories. We used triangulation and iterative data analysis to identify topics concerning laboratory business models and practices. RESULTS: We characterized laboratories as full-service (FSL), for-profit germline (PGL), and not-for-profit germline (NGL). Relying on existing payer contracts is a key characteristic of the FSL business models. FSLs focus on high-volume germline tests with evidence of clinical utility that have reimbursable codes. In comparison, a key business model characteristic of PGLs is direct patient billing facilitated by commodity-based pricing made possible by investors and industry partnerships. Client billing is a key business model characteristic of NGLs. Because many NGLs exist within academic settings, they are challenged by their inability to optimize laboratory processes and billing practices. CONCLUSION: Continued availability of, and access to germline testing will depend on the financial success of laboratories; organizational characteristics of laboratories and payers; cultural factors, particularly consumer interest and trust; and societal factors, such as regulation and laws surrounding pricing and reimbursement.

ADMIXPIPE: population analyses in ADMIXTURE for non-model organisms.

BACKGROUND: Research on the molecular ecology of non-model organisms, while previously constrained, has now been greatly facilitated by the advent of reduced-representation sequencing protocols. However, tools that allow these large datasets to be efficiently parsed are often lacking, or if indeed available, then limited by the necessity of a comparable reference genome as an adjunct. This, of course, can be difficult when working with non-model organisms. Fortunately, pipelines are currently available that avoid this prerequisite, thus allowing data to be a priori parsed. An oft-used molecular ecology program (i.e., STRUCTURE), for example, is facilitated by such pipelines, yet they are surprisingly absent for a second program that is similarly popular and computationally more efficient (i.e., ADMIXTURE). The two programs differ in that ADMIXTURE employs a maximum-likelihood framework whereas STRUCTURE uses a Bayesian approach, yet both produce similar results. Given these issues, there is an overriding (and recognized) need among researchers in molecular ecology for bioinformatic software that will not only condense output from replicated ADMIXTURE runs, but also infer from these data the optimal number of population clusters (K). RESULTS: Here we provide such a program (i.e., ADMIXPIPE) that (a) filters SNPs to allow the delineation of population structure in ADMIXTURE, then (b) parses the output for summarization and graphical representation via CLUMPAK. Our benchmarks effectively demonstrate how efficient the pipeline is for processing large, non-model datasets generated via double digest restriction-site associated DNA sequencing (ddRAD). Outputs not only parallel those from STRUCTURE, but also visualize the variation among individual ADMIXTURE runs, so as to facilitate selection of the most appropriate K-value. CONCLUSIONS: ADMIXPIPE successfully integrates ADMIXTURE analysis with popular variant call format (VCF) filtering software to yield file types readily analyzed by CLUMPAK. Large population genomic datasets derived from non-model organisms are efficiently analyzed via the parallel-processing capabilities of ADMIXTURE. ADMIXPIPE is distributed under the GNU Public License and freely available for Mac OSX and Linux platforms at: https://github.com/stevemussmann/admixturePipeline .

Perspectives of US private payers on insurance coverage for pediatric and prenatal exome sequencing: Results of a study from the Program in Prenatal and Pediatric Genomic Sequencing (P3EGS).

PURPOSE: Exome sequencing (ES) has the potential to improve management of congenital anomalies and neurodevelopmental disorders in fetuses, infants, and children. US payers are key stakeholders in patient access to ES. We examined how payers view insurance coverage and clinical utility of pediatric and prenatal ES. METHODS: We employed the framework approach of qualitative research to conduct this study. The study cohort represented 14 payers collectively covering 170,000,000 enrollees. RESULTS: Seventy-one percent of payers covered pediatric ES despite perceived insufficient evidence because they saw merit in available interventions or in ending the diagnostic odyssey. None covered prenatal ES, because they saw no merit. For pediatric ES, 50% agreed with expanded aspects of clinical utility (e.g., information utility), and 21% considered them sufficient for coverage. For prenatal ES, payers saw little utility until in utero interventions become available. CONCLUSION: The perceived merit of ES is becoming a factor in payers' coverage for serious diseases with available interventions, even when evidence is perceived insufficient. Payers' views on ES's clinical utility are expanding to include informational utility, aligning with the views of patients and other stakeholders. Our findings inform clinical research, patient advocacy, and policy-making, allowing them to be more relevant to payers.

Contrasting signatures of introgression in North American box turtle (Terrapene spp.) contact zones.

Hybridization occurs differentially across the genome in a balancing act between selection and migration. With the unprecedented resolution of contemporary sequencing technologies, selection and migration can now be effectively quantified such that researchers can identify genetic elements involved in introgression. Furthermore, genomic patterns can now be associated with ecologically relevant phenotypes, given availability of annotated reference genomes. We do so in North American box turtles (Terrapene) by deciphering how selection affects hybrid zones at the interface of species boundaries and identifying genetic regions potentially under selection that may relate to thermal adaptations. Such genes may impact physiological pathways involved in temperature-dependent sex determination, immune system functioning and hypoxia tolerance. We contrasted these patterns across inter- and intraspecific hybrid zones that differ temporally and biogeographically. We demonstrate hybridization is broadly apparent in Terrapene, but with observed genomic cline patterns corresponding to species boundaries at loci potentially associated with thermal adaptation. These loci display signatures of directional introgression within intraspecific boundaries, despite a genome-wide selective trend against intergrades. In contrast, outlier loci for interspecific comparisons exhibited evidence of being under selection against hybrids. Importantly, adaptations coinciding with species boundaries in Terrapene overlap with climatic boundaries and highlight the vulnerability of these terrestrial ectotherms to anthropogenic pressures.

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

Balloon-assisted remote external iliac artery endarterectomy: A safe and durable technique for the treatment of iliac artery occlusive disease.

BACKGROUND: Historically, the treatment of iliac artery occlusive disease required a surgical bypass usually consisting of an aortobifemoral bypass or an iliofemoral bypass. With the advent of balloon angioplasty and stenting, these procedures are frequently replaced with endovascular options. However, the treatment of diffuse occlusive disease of the external iliac artery (EIA) using balloon angioplasty and/or stenting does not carry a favorable long-term patency rate. Remote endarterectomy of the EIA using ring dissectors with balloon assistance provides a novel, controlled, safe, and durable treatment of the diseased and/or occluded EIA. METHODS: A retrospective review over the past 6 years was performed at our institution identifying patients treated with balloon-assisted remote endarterectomy of the EIA by the current five practicing vascular surgeons. The technique involves exposure of the ipsilateral common femoral artery. With nonocclusive disease, direct access into the common femoral artery is performed, a wire is traversed through the diseased EIA, and a balloon is inflated at the origin of the vessel providing hemostasis and control. A femoral endarterectomy is performed, and a ring dissector is passed over the endarterectomized material including the wire and balloon catheter and advanced remotely through the EIA up to the balloon. The balloon is briefly deflated, repositioned within the ring dissector, and reinflated, thus cutting the plaque. This allows for retraction of the inflated balloon and cutter, removing the endarterectomized core plaque. The procedure is similar for the treatment of an occluded EIA, but wire access across the occluded vessel is normally achieved with contralateral access. In both cases, the balloon provides control and hemostasis and is critically important in the rare treatment of vessel rupture. RESULTS: A total of 101 vessels were treated in 97 patients. The procedure was successful in 98 vessels (97%) with failure related to vessel rupture requiring conversion to an iliofemoral bypass. The estimated patency rate at three years was 94% with a median follow-up of 20 months. Restenosis/occlusion in four patients seemed to be related to a severe sclerotic response. The EIA was occluded 32% of the time. The common iliac artery (CIA) was diseased requiring angioplasty and stenting 29% of the time and a stent was placed at the transition zone between endarterectomized vessel and nontreated proximal most EIA or distal most CIA 58% of the time. There were no perioperative deaths. CONCLUSIONS: Balloon-assisted remote endarterectomy of the diffusely diseased and/or occluded EIA is a safe and durable option. It precludes the need for a prosthetic conduit and the risk of associated infection. It also involves a single groin incision and negates the need for retroperitoneal exposure of the CIA.